Histamine measuring apparatus and a histamine measuring method

ABSTRACT

Histamine may be quantitatively measured by providing the steps of: holding in a recess formed at the bottom of a vessel an oocyte that expresses histamine receptors, inserting first and second electrodes into the oocyte, measuring the membrane potential of the oocyte by means of said first electrode to stabilize the membrane potential of said oocyte at a predetermined level by driving a current through second electrode inserted into said oocyte by means of a circuitry for clamping membrane potential of oocyte, infusing a sample into a fine reacting tube having an antigen immobilized on the inner surface thereof together with some buffer solution to promote a histamine releasing reaction, transferring the solution containing histamine released in the fine reacting tube to the vessel to make contact with said oocyte in the vessel, detecting an electric response of oocyte caused by the contact with said solution, and determining the concentration of histamine released by said histamine releasing reaction in said fine reacting tube. The whole blood or mast cell suspension may be used as a sample without pretreatment.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is directed to a histamine measuringapparatus and a histamine measuring method for quantitatively measuringhistamine present in bloods or mast cells suspensions.

[0003] 2. Description of the Prior Art

[0004] Histamine releasing test is a quantitative analysis of histamineextracellularly released by stimulating leucocytes (white blood cells)in the blood or mast cells in the mucosa and promoting releasinghistamine contained therein outside the cells. The histamine releasetest has been reported to be a method useful in identifying someallergens in some allergic disorders and diseases (c.f., Prior Art 1:Tabe, kazuaki: histamine release test—diagnosis of allergies, SRL HOKAN,vol 21, pp. 17-22 (1997).

[0005] The known measurements of freed histamine (histamine released infree state) includes, for example, a method using fluorescent HPLC(fluorescent high performance liquid chromatography) to purify thehistamine to react it with a fluorescent reagent in order to measure theamount of fluorescence emitted from the fluorescent reagent reacted withthe histamine (c.f., Prior Art 2: Japanese Patent Laid-Open No.H6-331619), a method using glass fibers to purify the freed histamine toreact it with a fluorescent reagent in order to measure the amount offluorescence emitted from the fluorescent reagent coupled with the freehistamine (c.f., Prior Art 3: Japanese Patent Laid-Open No. H10-170514,Japanese Patent Laid-Open No. 10-62415), competitive immunoassayscommercially available from ICN Pharmaceuticals or immunotech, and anELISA (c.f., Prior Art 4).

SUMMARY OF THE INVENTION

[0006] In general, the measurement of histamine may require:

[0007] (1) direct analysis of histamine without using labeling thereof;

[0008] (2) direct quantitative measurement of histamine withoutpretreatment;

[0009] (3) quick delivery of results; and

[0010] (4) specificity to the histamine.

[0011] Prior Art methodologies as have been described above are allindirect, quantitative measurement methods of histamine, which isolatethe histamine from a sample, and label the isolated histamine with afluorescent reagent to measure the labeled histamine reacted with afluorescent reagent. All of the Prior Art methodologies requires complexpretreatment such as purification of samples, isolation of histamine,and labeling of histamine with a fluorescent reagent, and also requiresfor hours to obtain a quantitative results of histamine analysis.

[0012] The primary object of the present invention is in general toprovide a histamine measuring apparatus and a histamine measuringmethod, which may satisfy the requirements listed above of histaminemeasurement.

[0013] In accordance with the histamine quantitative analyzing apparatusand method of the present invention, histamine quantitative analysiswill be achieved by:

[0014] providing cells with histamine receptor being expressed;

[0015] adding a sample having been stimulated by an antigen to the cellsexpressing the receptor; and

[0016] detecting the resulting electrical response of cells.

[0017] For example, a predetermined amount of antigen will be added to asample of collected whole blood, the sample with antigen will beincubated for 10 to 30 minutes at 37° C. to promote an allergic reaction(antigen challenge) therebetween to release the histamine into theplasma. It may be preferable to gently shake the blood sample whilepromoting the allergic reaction. The period of time of reactiondescribed here is indicated by way of example. The reaction time mayvary depending on the optimized allergic reaction.

[0018] Examples of sample include, whole blood specimen, leucocytes inthe blood, and mast cell suspensions in which the mast cells of mucosaltissue are cultured.

[0019] An allergic reaction may be invoked in general by the binding ofan allergen with a corresponding IgE present in the cell membrane ofcells contributing to the allergic reaction such as mast cells,basophils, in the sample blood. The binding may trigger a reaction ofreleasing a relevant chemical mediator (chemical transmitter) such ashistamine in the cells contributing to the allergy reaction. In thedescription hereinbelow, the reaction of releasing histamine will bereferred to as “histamine release (releasing) reaction.”

[0020] The concentration (A) of histamine extracellularly released bythe histamine releasing reaction may be determined by using acalibration curve thereof. The calibration curve maybe obtained bystimulating cells with the histamine receptor preliminary expressed bymeans of a plurality of known concentrations of histamine to detect thecellular electric responses at respective concentration of histamine andmay be expressed as a relation between a plurality of knownconcentrations of histamine and detected electric responses.

[0021] The ratio of histamine release a (%) may be determined by

α=100×(A−C)/B

[0022] where B is a histamine concentration given by the quantitativemeasurement of histamine released from the sample after freezing andthawing the sample having stimulated by the antigen, C is aconcentration of free histamine released in non stimulated state byadding some buffer solution instead of the antigen to the sample.

[0023] The concentration of released histamine (C) designates to areleased histamine concentration by the stimulation with the buffer,which is so-called “concentration for the correction of backgroundlevel”.

[0024] The apparatus and method in accordance with the present inventionallows the histamine released in the sample to be directly andquantitatively analyzed by using cells with histamine receptorsexpressed. The cells expressing histamine receptors may identifyspecifically and directly the histamine, even in case in whichderivatives of histamine or histamine-like compounds are present in thesample, or in case in which there is a trace of histamine contained inthe sample. The apparatus and method in accordance with the presentinvention, accordingly, may allow direct quantitative analysis ofhistamine to be performed in a shorter time, with no complexpretreatment of samples which may require long time.

[0025] An exemplary configuration of the present invention will besummarized below. The oocyte expressing the histamine receptors is heldin the recess formed at the bottom of a vessel. A first electrode and asecond electrode will be inserted into the oocyte to determine themembrane potential of oocyte by the first electrode, and then maintainthe potential of oocyte membrane to a predetermined level by flowingelectric current through the second electrode, by means of a circuitryfor maintaining the potential of oocyte membrane. A sample will be flewthrough together with some buffer into a fine reacting tube with anantigen fixed on the inner surface of tube wall to promote the histaminereleasing reaction. Thereafter the solution containing free histaminewill be flew through a flowing tube into the vessel to make contact withthe oocyte in the vessel. The electric response of the oocyte caused bythe contact with the solution will be detected by the potentialmaintainer (clamping) circuit in order to determine the concentration offree histamine released by the histamine releasing reaction. The wholeblood, or suspension may be used as the sample, without the need for anypretreatment.

[0026] In accordance with the present invention, the concentration ofhistamine may be determined in a shorter time, without the need for anypretreatment, so that the process steps for quantitative analysis, thenumber and amount of reagents may be reduced to minimum and the cost andthe time needed for the measurement may be significantly saved.

[0027] In the following description an oocyte of Xenopus lavis (Africanclawed frog) will be used for the cell expressing the histaminereceptors, byway of example. It should be understood that the presentinvention may be applicable by using any other types of cell.

[0028] Also in the following description of preferred embodiments, anexemplary case will be described in which the electric response of thecell with histamine receptors expressed will be detected by identifyinghistamine with the histamine receptors expressed on the cell membraneand by measuring the change in the membrane potential along with theopen and close of chloride ion channel caused by the intracellularsignal transduction. It is to be understood that the preferredembodiments described herein are presented and described for the purposeof illustrating the principle of the present invention and are notintended to be limiting. For example, it should be recognized that thepresent invention may be achieved by using the cellular response derivedfrom the receptor stimulation of any other kinds such as those describedin the Japanese Patent Laid-Open No. H11-083785 (Prior Art 5) or byusing any other detecting methods.

[0029] Additional objects and advantages of the invention will be setforth in part in the description which follows and in part will beobvious from the description, or may be learned by practice of theinvention. The objects and advantages of the invention may be realizedand attained by means of the instrumentalities and combinationsparticularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 shows a schematic diagram illustrating an apparatus inaccordance with one preferred embodiment of the present invention, whichis a histamine measuring apparatus for quantitatively analyzing thehistamine by using the oocyte with histamine receptors expressed;

[0031]FIG. 2 shows a schematic diagram illustrating an exemplaryanalysis of histamine concentration and electric response of the oocyteboth obtained by the apparatus shown in FIG. 1;

[0032]FIG. 3A shows a schematic diagram illustrating an exemplaryanalysis of electric response of oocyte, induced by the blood samplecontaining histamine, and obtained by using the apparatus shown in FIG.1;

[0033]FIG. 3B shows a schematic diagram illustrating an exemplaryanalysis of electric response of oocyte, induced by the blood samplecontaining no histamine, and obtained by using the apparatus shown inFIG. 1;

[0034]FIG. 4 shows a protocol of histamine analysis method in accordancewith a preferred embodiment of the present invention;

[0035]FIG. 5 shows a result of histamine analysis using a whole bloodsample, obtained by using the apparatus shown in FIG. 1; and

[0036]FIG. 6 shows a schematic diagram of an apparatus for measuringhistamine in accordance with another preferred embodiment of the presentinvention, illustrating the overview of apparatus for quantitativelyanalyzing histamine by using the oocyte with histamine receptorsexpressed and a whole blood sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In the following description of the preferred embodiments, thehistamine measuring apparatus and the histamine measuring method inaccordance with the present invention will be described in greaterdetails with reference to the quantitative analysis of histamine in thewhole blood sample by using the oocyte with histamine receptorsexpressed.

[0038] A whole blood sample stimulated by the antigen will be added tothe oocyte with histamine receptor expressed to detect the resultingelectric response of the oocyte. A predetermined amount of antigen willbe added to the collected whole blood sample (some buffer may beintroduced if the whole blood sample is too viscous), and the wholeblood sample and the antigen will be incubated at a temperature rangingfrom 30° C. to 45° C. for 10 to 30 minutes for promoting the allergyreaction therebetween in order to release histamine into the plasma.

[0039] The concentration of histamine (A) released into the plasma fromthe blood cells in the sample caused by the histamine releasing reactionmay be determined by means of a calibration curve. The calibration curvemay be obtained by stimulating cells with the histamine receptorpreliminary expressed by means of a plurality of known concentrations ofhistamine to detect the cellular electric responses at respectiveconcentration of histamine and may be expressed as a relation between aplurality of known concentrations of histamine and detected electricresponses.

[0040] The ratio of histamine release a (%) may be determined by

α=100×(A−C)/B

[0041] where B is a histamine concentration given by the quantitativemeasurement of histamine released from the cells in the sample bloodafter freezing and thawing the sample stimulated by the antigen, C is aconcentration of released histamine in non stimulated slate by addingsome buffer instead of the antigen into the blood sample.

[0042] The concentration of released histamine in non-stimulation Cdesignates to the concentration of free histamine released by thestimulation of buffer, i.e., so-called “concentration of correction ofbackground”.

[0043] The histamine analysis as have been described above may becarried out within approximately 20 to 50 minutes, including thecalibration measurement for determining the calibration curve to beused. In case in which the calibration curve has been determined inadvance, then the histamine measurement will be completed at most in 30seconds for each sample.

[0044] In this preferred embodiment of the apparatus and method inaccordance with the present invention, quantitative measurement ofhistamine released from the cells in the whole blood to the plasma maybe directly carried out. If the histamine derivatives or histamine-likecompounds are present in the cells or plasma of whole blood sample, orif the amount of histamine to be released into the plasma from the cellsof whole blood is very low, the oocyte with histamine receptorsexpressed will specifically and directly identify the histamine.Accordingly the apparatus and method for measuring histamine inaccordance with the present invention may directly and quantitativelyanalyze the released histamine from the whole blood cells to the plasmawithout the need for any pretreatment of whole blood sample.

[0045] Since the apparatus and method of the preferred embodiment inaccordance with the present invention may detect the histamine by usingthe oocyte with histamine receptors expressed the electric response ofthe oocyte caused by the physiological reaction induced at the time whenthe oocyte identifies the presence of histamine may be directly detectedas an electric signal. The physiological reaction of oocyte is known tobe occurred within milliseconds, thus the time required for detectingthe histamine may be within one second.

[0046] The apparatus and method for measuring histamine of the preferredembodiment in accordance with the present invention may perform aquantitative measurement in significantly shorter time than any otherPrior Art techniques requiring at least one hour for the analysis.

[0047] A detailed description of some preferred embodiments embodyingthe present invention will now be given referring to the accompanyingdrawings.

[0048]FIG. 1 shows a schematic diagram Illustrating an apparatus inaccordance with one preferred embodiment of the present invention, whichis a histamine measuring apparatus for quantitatively analyzing thehistamine by using the oocyte with histamine receptors expressed.

[0049] In this embodiment the oocyte of Xenopus lavis (African ClawedFrog) will be used. The unfertilized oocytes (eggs) of Xenopus lavis aresubjected to be injected mRNA of histamine receptor. These oocytes willbe incubated for two or three days in a culture medium so as to causehistamine receptors to be expressed. The histamine receptors will beactive within the cell membrane of oocyte. When histamine binds to thehistamine receptors, the information will be transferred inside theoocyte through the mediator (second messenger) already present in theoocyte.

[0050] As the result of a plurality of chemical mediation inside theoocyte, intracellular calcium ions are raised in the oocyte, opening thechloride ion channel of the oocyte cell membrane. This reaction willcause the change in potential of cell membrane inside and outside theoocyte.

[0051] In the present preferred embodiment of the present invention thechange in the electric response of oocyte before and after the bindingof histamine to the histamine receptors, namely, the change inpotentials between the inside and the outside of oocyte cell membrane,will be detected as the change in electric current response.

[0052] In the present preferred embodiment of the present invention amethod of whole cell clamp will be used in which a feedback circuitrywill be used, which may flow current through the oocyte in the directionof suppressing the difference of membrane potential at the moment ofchange in potential of membrane of oocyte to maintain the potential to apredetermined constant holding potential.

[0053] As shown in FIG. 1, at the recess 16 (not shown) at the bottom ofa vessel 1 fulfilled with buffer solution 23 for the oocyte, an oocyte 2with the histamine receptors expressed will be held. The fine tips ofglass electrodes 3 and 4, filled with KCl of 3 mole, and containing anAg wire coated with AgCl will be inserted into the oocyte 2. After theinsertion, each of the tips of glass electrodes 3 and 4 will be fixedlyheld by an apparatus for holding electrodes, which is not shown in FIG.1, but is provided correspondingly for respective electrode.

[0054] The potential of the glass electrode 3 will be transmitted to thedifferential amplifier 6 and the recorder 7 and recorded therein as thedifference in potential to an external electrode 5. The glass electrode3 may be an electrode for measuring the potential in the membrane ofoocyte, while the glass electrode 4 may be used for maintaining themembrane potential of the oocyte to a predetermined constant level of−60 mV.

[0055] The differential amplifier 6 will apply the differential currentbetween the signal transferred from the glass electrode 3 to thedifferential amplifier 6 and the holding potential in the whole cellclamping method to the oocyte 2 through the glass electrode 4. In thismanner, when the histamine 8 will be dropped onto the oocyte 2 with thepotential of cell membrane held at a constant level of −60 mV, by meansof for example a micro-syringe or a pipette 9, the electric response(current response 10) of the oocyte 2 may be obtained. The currentresponse 10 will be described in greater details below.

[0056]FIG. 2 shows a schematic diagram illustrating an exemplaryanalysis of histamine concentration and electric response of the oocyteboth obtained by the apparatus shown in FIG. 1. The abscissa is theknown concentration of histamine (nM) of the samples. The ordinate isthe amount of change in potential between the inside and outside of theoocyte before and after the binding of histamine to the histaminereceptors, measured as the change of current (μA)

[0057] As shown in FIG. 2, it is clear that the current change is inpositive correlation with the concentration of histamine in the range ofhistamine concentration from 20 nM to 200 nM. This correlation(calibration curve) may be used for measuring a sample containinghistamine at an unknown concentration by using the apparatus of thepresent invention shown in FIG. 1 so as to determine the unknownconcentration of histamine.

[0058]FIG. 3A and FIG. 3B show schematic diagrams illustrating anexemplary analysis of electric current response of the oocyte, inducedby the whole blood sample containing histamine (FIG. 3A) and by theblood sample containing no histamine (FIG. 3B) and obtained by using theapparatus shown in FIG. 1.

[0059] As shown in FIG. 3A, when at the moment as shown by the verticalarrow, the whole blood sample containing histamine is added to theoocyte expressing histamine receptors, the current response will bereached at the maximum value within one second, then will decreasegradually together with the elapsed time to disappear approximately 30seconds after.

[0060] On the other hand, as shown in FIG. 3B, when the whole bloodsample containing no histamine is added to the oocyte expressinghistamine receptors at the moment as shown by the vertical arrow, nochange in current response will be observed. This indicates that thechange in current response as shown in FIG. 3A is based on the reactionbetween the histamine and histamine receptors, rather than the change orresponse caused by the impurity of whole blood sample. When applying themaximum value of the current response shown in FIG. 3A to thecorrelation (calibration curve) shown in FIG. 2, it can be seen thatthis maximum value of the current response may be caused by thehistamine of concentration of approximately 100 nM. The outlinedvertical arrow shown in FIG. 3A indicates the change in current beforeand after adding a whole blood sample containing histamine to the oocyteexpressing histamine receptors.

[0061] As can be seen from the foregoing description, in accordance withthe present invention, a whole blood sample may be used for quantitativeanalysis of histamine without needs for isolation, generator andlabeling. As have been described above, by measuring the change inmembrane potential as the change in current passing therethrough, alongwith the opening and closing of chloride ion channel caused byintracellular chemical mediator after identifying histamine by an oocyteexpressing histamine receptors, the measurements for quantitativeanalysis of histamine may be obtained within one second after the oocyteidentifies histamine.

EMBODIMENTS

[0062] First Embodiment

[0063] By stimulating oocytes expressing histamine receptors with aplurality of known concentrations of histamine to detect the change incurrent as have been described above, the correlation (calibrationcurve) between the change in current and the concentrations of histamineas shown in FIG. 2 may be predetermined.

[0064]FIG. 4 shows a protocol of histamine analysis method in accordancewith a preferred embodiment of the present invention. The protocol willbe described below in greater details with reference to FIG. 4:

[0065] (1) A whole blood sample 11 will be collected from vein of ahuman elbow, using heparin as anticoagulant.

[0066] (2) A predetermined amount of antigen 12 such as cedar pollenwill be added to the collected whole blood sample 11.

[0067] (3) The whole blood sample 11 will be incubated for approximately30 minutes at 37° C. to promote histamine releasing reaction between thewhole blood and antigen. Due to the allergy reaction basophils 13 in thewhole blood sample will be stimulated and will release histamine 8;

[0068] (4) The whole blood sample 11 having stimulated by the antigen 12will be sampled by means of a micro-syringe or a pipette.

[0069] (5) The sample will be added to the oocyte expressing histaminereceptors.

[0070] (6) The change in electric response of the oocyte will bemeasured by means of the apparatus shown in FIG. 1 to determine thechange in current as have been described above.

[0071] (7) From the current change value, the concentration of histamine(A) in the whole blood sample 11 having stimulated by the antigen 12 maybe determined by referring to she predetermined correlation (calibrationcurve).

[0072] The releasing rate of histamine α (%) may be determined by

α=100×(A−C)/B

[0073] where B is a histamine concentration given by the quantitativemeasurement of histamine released from the cells in the sample wholeblood after freezing and thawing the sample having stimulated by theantigen, C is a concentration of free released histamine in nonstimulated state by adding the buffer solution 23 instead of the antigeninto the blood sample.

[0074] The concentration of free histamine released in non-stimulation Cdesignates to the concentration of free histamine released by thestimulation of buffer, i.e., so-called a “concentration of correction ofbackground”.

[0075] The time required for a quantitative analysis of histamine inaccordance with the first preferred embodiment will be approximately 30minutes, which is significantly faster when compared with 3 hours neededfor the method described in the Prior Art 3 above and 4 hours needed forthe method described in the Prior Art 4 above, resulting in a saving oftime.

[0076] The procedure of the method of measuring histamine in accordancewith the first preferred embodiment of the present invention ischaracterized in that the collected sample may be used for quantitativeanalysis without need of any pretreatment of whole blood, withsignificantly fewer steps of processing as compared with any of PriorArt. The procedure of the method in accordance with the presentinvention requires only histamine of known concentration at the time ofmaking a calibration curve, and no other reagents are needed, allowingthe analytical cost to be reduced.

[0077] Second Embodiment

[0078]FIG. 5 shows an example of results of histamine analysis usingwhole blood samples (5 samples), obtained by using the apparatus shownin FIG. 1. The cedar pollen was used as antigen. FIG. 5 shows theresults of measurement of histamine, A (concentrations of histamine inthe whole blood samples stimulated by the cedar pollen), B(concentrations of histamine quantitatively determined from thehistamine released from the cells of whole blood samples by freezing andthawing the whole blood samples stimulated by the cedar pollen), C(concentrations of freely released histamine in non stimulated statederived from the cells in the whole blood sample by adding the buffersolution 23 thereto instead of the cedar pollen antigen) and α (%)(histamine releasing rate), each determined for respective sample inaccordance with the procedure similar to that of the first embodiment ashave been described above.

[0079] In the histamine-releasing test in general with respect to bloodsamples, when the histamine-releasing rate of a sample is higher than orequal to 10% when stimulating with a specific allergen, the sample maybe considered to be positive in the allergy reaction. In the exemplaryembodiment shown in FIG. 5, the samples 1 and 3 are to be consideredthat the allergy reaction for the cedar pollen has been occurred. Theseresults were consistent with -he results of conventional HRT (HistamineRelease Test) method as well as the subjective symptom of examinees.

[0080] Third Embodiment

[0081]FIG. 6 shows a schematic diagram of an apparatus for measuringhistamine in accordance with another preferred embodiment of the presentinvention, illustrating the overview of an apparatus for quantitativelyanalyzing histamine by using the oocyte with histamine receptorsexpressed and a whole blood sample.

[0082] The apparatus shown in FIG. 6 is consisted of, in addition to thecomponents of the apparatus shown in FIG. 1, a pouring tube 14 forpouring the buffer solution 23 into the vessel 1 with the assistance ofa pump and the like, a draining tube 15 for draining excessive buffersolution 23 from the vessel 1 with the assistance of a pump and thelike, and a draining tube 20 for purging the solution contained in thevessel 1 with the assistance of a pump and the like each time when thehistamine analysis of a sample is completed. It should be noted that inFIG. 6 the external electrode 5, the differential amplifier 6, thegrounding wire of differential amplifier 6, the recorder 7, the electricresponse of oocyte 10 are omitted for the sake of simplification.

[0083] The buffer solution 23 for oocyte 2 will be infused to the vessel1 through the pouring cube 14 in excess of the liquid level detectorportion of liquid level meter (not shown in FIG. 6) disposed inside thevessel. Then the driving apparatus of drain pump will be driven based onthe output from the level detector until the level of buffer 23 willreach to the level of the level detector sensing portion in order todrain the excessive buffer solution 23 from the vessel 1 through thedraining tube 15.

[0084] Thereafter, if for some reason or another the level of buffersolution 23 contained in the vessel 1 is decreased or increased, thedecrease of buffer solution 23 will be detected by the level detectorsensor portion and the driving apparatus of drain pump will be driven inaccordance with the output of the level detector to automatically add anamount of buffer solution 23 through the pouring tube 14 into the vessel1 in order to maintain the level of buffer solution 23 at apredetermined level. In this manner a predetermined constant amount ofbuffer solution 23 sufficient for dipping the oocyte 2 will be held inthe vessel 1.

[0085] As shown in FIG. 6, in the recess 16 at the bottom of the vessel1 filled with the buffer solution 23 for oocyte 2, a Xenopus oocyteexpressing histamine receptors 2 will be immersed in a static manner. Assimilar to the apparatus shown in FIG. 1, the fine tips of glasselectrode 3 and glass electrode 4 will be inserted into the Xenopusoocyte 2. The potential appeared in the glass electrode 3 will betransmitted to the differential amplifier 6 not shown in FIG. 6 as thedifferential potential from the external electrode 5. The differentialamplifier 6 receiving the potential signaled from the glass electrode 3will generate the current difference between the glass electrode 3signal and the fixed potential in the whole cell clamping method andapply it to the Xenopus oocyte 2 through the glass electrode 4. Themembrane potential of the oocyte will be accordingly held at apredetermined constant level of −60 mV.

[0086] Then the whole blood sample 1 prepared in accordance with theprocedure shown in FIG. 4 will be added gently over the Xenopus oocyte 2the membrane potential of which is held at −60 mV by means of aninstrument such as micro-syringe or pipette 9. The whole blood sample 11added to the vessel over the oocyte contains the histamine 8 released bythe histamine releasing reaction, antigens 12, basophils 13, plasma andthe like. The change of current before and after the histamine 8 isbound to the histamine receptors will be measured. As have beendescribed in the foregoing discussion, the concentration of histamine 8released from the cells in the whole blood may be determined from thechange of current by predetermining a calibration curve.

[0087] Another configuration of apparatus for adding the prepared wholeblood sample 11 over the Xenopus oocyte 2 without using a micro-syringeor pipette 9 will be described below in greater details. As shown inFIG. 6, a fine reacting tube (glass capillary) 17 may be provided to heapparatus for carrying out therein the histamine releasing reaction inthe whole blood sample, such that step (3) of the procedure shown inFIG. 4 will be completed within the fine reacting tube 17. On the innersurface of the fine reacting tube 17 the antigen 12 such as cedar pollenand the like are immobilized.

[0088] A temperature controlling device (heater) 18 configured so as tosurround the outer surface of tubing wall of the fine reacting tube 17will control the temperature of fine reacting tube 17 at a temperatureranging from 30° C. to 45° C. for promoting the histamine releasingreaction. 5 to 20 mL of whole blood sample will be directly poured intothe fine reacting tube 17 held at 37° C. via the tubing of left handside of a cock 22 in the Figure and the sample will be held therein for30 minutes to initiate and promote the histamine releasing reaction.

[0089] Once the histamine releasing reaction in the fine reacting tube17 has been completed, the temperature inside the fine reacting tube 17will be decreased back to the room temperature and then a cock 19 willbe opened so that the blood sample solution containing the histamine 9released by the histamine releasing reaction, antigen 12, basophils 13,plasma and the like will be transferred to the surface of the Xenopusoocyte 2 held at the recess 16 of the bottom of vessel 1 through thecock 19 and flowing tube 21. Through the fine tip of the flowing tube21, the solution containing histamine 8 and the like will be added overthe Xenopus oocyte 2. After transfer the cock 19 may be closed.

[0090] The cross-sectional form of the flowing tube 21 is round and thecube 21 is bent at the proximity of its tip. The flowing tube 21 will beinserted to an opening provided on the sidewall of the vessel 1 in roundcross-sectional shape. The axis of the opening aligns to the projectionline extendingly projected to the bottom of vessel 1, the axis passingthrough the center of recess 16. The flowing tube 21 may be movable inrotative and translatory manner within the opening. The relativeposition of the tip of flowing tube 21 with the surface of oocyte heldon the recess 16 may be adjusted by moving the flowing tube 21 inrotative or translatory direction as described above.

[0091] After the adjustment of relative position, the sample solutioncontaining the histamine 8, antigen 12, basophils 13, plasma and thelike will be gently add the Xenopus oocyte 2 having the membranepotential held at −60 mV by controllably flowing the buffer solution 23or sterile gas from the tubing of left hand side of the cock 22 with theflow rate being controlled.

[0092] As have been described above, by measuring the change in currentbefore and after the histamine 8 is bound to the histamine receptors ofoocyte and using the predefined calibration curve, the concentration ofhistamine 8 released from the cells in the whole blood sample may bedetermined.

[0093] When the measurement of change in current has been completed, theglass electrodes 3 and 4 may be drawn from the oocyte, and the solutionin the vessel 1 together with the oocyte may be drained through thedraining tube 20 by for example suction by means of a purge pump. Thenthe electrodes 3 and 4 may be moved in one side of recess on which theoocyte will be held. Some washing solution may be poured into the vessel1 through the pouring tube 14 to stir washing solution with a stirrernot shown in the figure in the vessel 1 to wash and rinse the vessel andthereafter the washing solution may be drained through the draining tube20. The above washing process will be repeated three to four times tocomplete washing step of the vessel.

[0094] Then, by opening the cock 19 to flow washing solution through thetubing of left hand side of the cock 22 for several times to wash andrinse the inside of the fine reacting tube 17, cock 19, and flowing tube21. Finally, the above washing process will be iterated for severaltimes to complete washing step of the apparatus. In the configurationusing the fine reacting tube 17, the quantitative analysis of theconcentration of histamine released from the cells in the whole bloodsample along with the histamine releasing reaction of whole blood may becontinuously carried out. More specifically, the sequential change ofhistamine releasing process may be allowed to measure in a contiguousmanner. In such a case the relative position of the fine tip of flowingtube 21 and the oocyte held in the recess 16 should be controlled by therotative and translative displacement as have been described above priorto analysis.

[0095] Then the sample will be directly injected through the tubing ofleft hand side of the cock 22 to the fine reacting tube 17 having theantigen 12 such as cedar pollen immobilized on the surface of inner wallthereof and held by the temperature controlling device (heater) 18 at atemperature in a range from 30° C. to 45° C. for promoting the histaminereleasing reaction. Immediately after that, the cock 19 will be openedto flow the buffer solution 23 held at a temperature approximately sameto that of the fine reacting tube 17 at slower rate through the tubingof left hand side of the cock 22.

[0096] A membrane having a number of micropores through which thehistamine 8 may be passed but the basophils 13 may not be provided forexample between the fine reacting tube 17 and the cock 19.

[0097] Although the present invention has been described in conjunctionwith several preferred embodiments thereof, it should be understood thatthese embodiments are disclosed by way of examples and the presentinvention is not to be limited thereto. At should be recognized thatmany changes and modifications may be made by those skilled in the artwithout departing from the true spirit and the scope of the presentinvention according to the appended claims.

What is claimed is:
 1. A histamine measuring apparatus forquantitatively analyzing the concentration of histamine, comprising: avessel having at the bottom thereof a recess for holding an oocyte whichexpresses histamine receptors; first and second electrodes for insertinginto said oocyte; a circuitry for measuring the potential of membrane ofsaid oocyte by means of said first electrode and for flowing a currentthrough said second electrode to said oocyte to maintain the membranepotential of said oocyte at a predetermined constant level; a finereacting tube with an antigen immobilized onto the inner surfacethereof; and flowing tubes for flowing a sample together with somebuffer solution into said fine reacting tube to promote a histaminereleasing reaction therein and for transferring the solution containingreleased histamine into said vessel to make contact with said oocyte insaid vessel; wherein said circuitry detects the electric response fromsaid oocyte caused by the contact with said solution in order todetermine the concentration of histamine released by the histaminereleasing reaction occurred in said fine reacting tube.
 2. A histaminemeasuring apparatus according to claim 1 , in which: said sample is awhole blood sample or mast cell suspension without pretreatment.
 3. Ahistamine measuring apparatus according to claim 1 , further comprising:a temperature controller device for controlling the temperature of saidfine reacting tube to a predetermined temperature level.
 4. A histaminemeasuring apparatus according to claim 1 , further comprising: atemperature controller device for controlling the temperature of saidfine reacting tube to a temperature ranging from 30° C. to 45° C.
 5. Ahistamine measuring apparatus according to claim 1 , further comprising:a tubing connected to said vessel for infusing buffer solution theretoand a tubing for purging the content of said vessel.
 6. A histaminemeasuring apparatus for quantitatively analyzing the concentration ofhistamine, comprising: a vessel having at the bottom thereof a recessfor holding an oocyte, which expresses histamine receptors; a finereacting tube with an antigen immobilized onto the inner surfacethereof; flowing tubes for flowing a sample together with some buffersolution into said fine reacting tube to promote a histamine releasingreaction therein and for transferring the solution containing releasedhistamine into said vessel to make contact with said oocyte in saidvessel; and a circuitry for detecting electric response of said oocytecaused by the contact with said solution; wherein said apparatusdetermines the concentration of histamine released by the histaminereleasing reaction occurred in said fine reacting tube.
 7. A histaminemeasuring apparatus according to claim 6 , in which: said sample is awhole blood sample or mast cell suspension without pretreatment.
 8. Ahistamine measuring apparatus according to claim 5 , further comprising:a temperature controller device for controlling the temperature of saidfine reacting tube to a temperature ranging from 30° C. to 45° C.
 9. Ahistamine measuring method for determining the concentration ofhistamine, comprising the steps of: holding in a recess at the bottom ofa vessel an oocyte that expresses histamine receptors; measuring thepotential of membrane of said oocyte by means of first electrodeinserted into said oocyte to stabilize the membrane potential of saidoocyte at a predetermined level by driving a current through secondelectrode inserted into said oocyte; infusing a sample into a finereacting tube having an antigen immobilized on the inner surface thereofto promote a histamine releasing reaction; transferring the solutioncontaining histamine released in said fine reacting tube to said vesselto make contact with said oocyte in said vessel; detecting an electricresponse of said oocyte caused by the contact with said solution; anddetermining the concentration of histamine released by said histaminereleasing reaction in said fine reacting tube.
 10. A histamine measuringmethod according to claim 9 , in which: said sample is a whole bloodsample or mast cell suspension without pretreatment.
 11. A histaminemeasuring method according to claim 9 , further comprising the step of:controlling the temperature of said fine reacting tube at apredetermined temperature level.
 12. A histamine measuring methodaccording to claim 9 , further comprising the step of: controlling thetemperature of said fine reacting tube to a temperature ranging from 30°C. to 45° C.
 13. A histamine measuring method according to claim 9 ,further comprising the step of: determine the concentration (A) ofhistamine released by said histamine releasing reaction in said finereacting tube by means of a predefined calibration curve obtained bystimulating said oocyte with a plurality of known concentrations ofhistamine to detect the electric response of said oocyte to predefinethe correlation between said electric responses and said plurality ofknown concentrations of histamine.
 14. A histamine measuring methodaccording to claim 9 , further comprising the step of: determining thehistamine releasing rate given by (A−C)/B where B is a histamineconcentration given by the quantitative measurement of histaminecontained in the cells in said sample and released by freezing andthawing the sample stimulated by the antigen, C is a concentration offree histamine released without stimulation after adding some buffersolution instead of the antigen into the sample.
 15. A histaminemeasuring method for determining the concentration of histaminecomprising the steps of: holding in a recess at the bottom of a vesselan oocyte that expresses histamine receptors; infusing a sample into afine reacting tube having an antigen immobilized on the inner surfacethereof to promote a histamine releasing reaction; transferring thesolution containing histamine released in said fine reacting tube tosaid vessel to make contact with said oocyte in said vessel; detectingan electric response of said oocyte caused by the contact with saidsolution; and determining the concentration of histamine released bysaid histamine releasing reaction in said fine reacting tube.
 16. Ahistamine measuring method according to claim 15 , in which: said sampleis a whole blood sample or mast cell suspension without pretreatment.17. A histamine measuring method according to claim 15 , furthercomprising the step of: controlling the temperature of said finereacting tube to a temperature ranging from 30° C. to 45° C.
 18. Ahistamine measuring method according to claim 15 , further comprisingthe step of: determine the concentration (A) of histamine released bysaid histamine releasing reaction in said fine reacting tube by means ofa predefined calibration curve obtained by stimulating said oocyte witha plurality of known concentrations of histamine to detect the electricresponse of said oocyte to predefine the correlation between saidelectric responses and said plurality of known concentrations ofhistamine.
 19. A histamine measuring method according to claim 15 ,further comprising the step of: determining the histamine releasing rategiven by (A−C)/B where B is a histamine concentration given by thequantitative measurement of histamine contained in the cells in saidsample and released by freezing and thawing the sample stimulated by theantigen, C is a concentration of free histamine released withoutstimulation after adding some buffer solution instead of the antigeninto the sample.